The well-studied biology of the T-even phages and their host,Escherichia coli, provides us with a unique opportunity for molecular analysis of newly-discovered self-splicing RNAs, by genetic dissection as well as comparative study. The goals of the proposed project include a fundamental structure-function analysis of self-splicing RNAs, as well as study of the generality and distribution of naturally occurring introns in this eubacterial system. Thse investigations will be greatly facilitated by powerful prokaryotic genetic strategies and by the availability of useful phage mutants and plasmid constructs. In addition to the intron in the thymidylate synthase (td) gene, two other group I introns were recently reported in T4 (Gott, J.M., Shub, D.A. and Belfort, M. Cell (1986) 47.81). One of these is within the nrdB gene encoding ribonucleoside diphosphate reductase subunit B, while the other is at an uncharacterized locus in the nrdC-gene 55 region of the T4 map. These two new introns are therefore available for analysis and comparison with one another,with the td intron, with eukaryotic group I introns and with other prokaryotic introns that may emerge from proposed studies. Primary sequence, secondary structure, the in vivo and in vitro splicing reactions and the key elements of the splicing process as determined by mutational analysis will be compared. Consensus structural elements and functional conservation on one hand, and the nature, distribution and phenotypes of splicing- defective mutations and second-site revertants on the other, will delineate critical structural and functional components of the group I splicing pathway. In addressing the generality of introns in this prokaryotic system we shall explore the existence of group I introns in E. coli and probe the occurrence of factor-dependent group I introns and of group II introns in T4. Further, the phages T2 and T6 provide a favorable system for comparison of the structure and distribution of introns in closely related organisms. Interest in this aspect of the work is heightened by our recent observations that indicate the variable occurrence of the different introns in the T-even phages, and that therefore suggest intron mobility. The overall study will thus address issues of generality, distribution, evolutionary history and possible mobility of introns as well as analyze the basic self-splicing process, in this genetically defined and manipulable prokaryotic model system.